Advances in vaccine development for Chlamydia trachomatis

Abstract Chlamydia trachomatis is the most prevalent bacterial sexually transmitted infection globally. Antibiotic treatment is highly effective, but infection is often asymptomatic resulting in most individuals going undetected and untreated. This untreated infection can ascend to the upper female genital tract to cause pelvic inflammatory disease, tubal factor infertility, and ectopic pregnancy. Chlamydia screening and treatment programs have failed to control this epidemic and demonstrate the need for an efficacious vaccine to prevent transmission and disease. Animal models and human epidemiological data reveal that natural immunity can provide partial or short-lived sterilizing immunity. These data further demonstrate the importance of eliciting interferon gamma (IFNγ)-producing cluster of differentiation 4 (CD4) T cells (Th1 and Th1/17 cells) that can likely synergize with antibody-mediated opsonophagocytosis to provide optimal protection. These studies have guided preclinical rational vaccine design for decades and the first Phase 1 clinical trials have recently been completed. Recent advances have led to improvements in vaccine platforms and clinically safe adjuvants that help provide a path forward. This review describes vaccine models, correlates of immunity, antigen and adjuvant selection, and future clinical testing for Chlamydia vaccine development.


Introduction
Chlam ydia trac homatis (CT) is a gr am-negativ e obligate intr acellular bacterium that infects the ocular and genital tract epithelium.Ser ov ars A, B, Ba, and C cause ocular tr ac homa, and sexuall y tr ansmitted genital infection and associated diseases are caused by ser ov ars D-K, with ser ov ars L1-L3 causing l ymphogr anuloma v ener eum (LGV).The de v elopmental cycle is made up of the elementary body (EB) form that attaches and infects the epithelial target cell and then differentiates into the reticulate body (RB) form that replicates in an intracellular inclusion.The RBs differ entiate bac k to infectious EBs, which are released from the host cell to begin another round of infection.Genital CT infection and associated diseases are a significant global health burden with ∼131 million new cases occurring each year (Newman et al. 2015 ).In the United States, > 1.8 million people are infected annually (Geisler et al. 2022 ) and direct treatment costs exceed $500 M (Owusu-Edusei et al. 2013 ).Antibiotic treatment is highly effective but because > 70% of infections are asymptomatic (Stamm 1999, Farley et al. 2003 ) , many infected persons go undetected and untreated.In women, CT infects the endocervix but can spread to the endometrium and oviducts to cause symptomatic ( ∼10%) or subclinical ( ∼25%) pelvic inflammatory disease (PID).Sequelae include c hr onic pelvic pain (30%), infertility (10%), and ectopic pregnancy (10%) (Haggerty et al. 2010 ).CT enhances the acquisition of other STIs and is an independent risk factor for cervical cancer (Chan et al. 2016 ).Prior CT infection increases the risk for fallopian tube carcinomas and epithelial ovarian cancer.In men, urethritis, e pidid ymitis, and orc hitis ar e the most common acute manifesta-tions (Mackern-Oberti et al. 2017 ), and while long term-sequelae are not associated with CT genitourinary infection in males, proctitis , prostatitis , and prostate cancer have been linked to CT, particularly in men who have sex with men (MSM) (Bryan et al. 2020 ).Also, among MSM, 10%-15% test positive for rectal Chlamydia at sexual health clinics and the US Centers for Disease Control and Pr e v ention r ecommends at least annual screening in MSM individuals engaging in rece pti ve anal sex (Khosropour et al. 2021 ).
LGV infections are also increasing and can result in highly debilitating c hr onic sequelae like bubo formation, fistulas , fibrosis , and rectal stenosis (Williamson and Chen 2020 ).Despite screening pr ogr ams, infection r ates continue to rise (Shannon and Klausner 2018 ).Barrier methods of contraception like condom use are effective but utilization rates are low (Bearinger et al. 2007 ).There is a clear and urgent need for a vaccine that combats this ongoing epidemic and associated complications, which will likely require immunization of both sexes to prevent transmission.

Models of CT genital infection
Chlamydiae infect a wide range of species, but productive infection is gener all y host-specific, with significant limitations to using CT in mice .CT sero var D shedding is detected for only 10 days in imm unologicall y normal mice after v a ginal inoculation (O'Connell et al. 2011 ), and the infection course is unaltered in mice lacking CD4 T cells (Morrison et al. 2011 ) suggesting CT deliver ed intr av a ginall y is insufficient for assessing ada ptiv e imm une r esponses.Tr anscervical inoculation of mice with human strains r esults in mor e pr oductiv e infections (Rajee v e and Siv adasan 2020 ), but bypasses the cervix, a significant mucosal and immunological barrier to the upper genital tract.Transcervical inoculation with human ser ov ars induces minimal oviduct pathology, presenting another limitation to this model.T hus , preclinical studies in mice fr equentl y use the natural mouse pathogen Chlamydia muridarum (CM) since CT is highly susceptible to murine IFN γ -induced GTPases (Nelson et al. 2005 ).Female mice inoculated v a ginall y with CM r eca pitulate imm une mediators of pr otection and fallopian tube pathology observed with se v er e CT infection in cisgender women.This pathology, specifically oviduct scarring and fibrosis with post-obstructive dilatation or hydrosalpinx, provides a robust model for vaccine testing.The female mouse model has been extensiv el y utilized in the field, while the male and male-to-female sexual transmission model has gained focus over the last 10 years (Sobinoff et al. 2015, O'Meara et al. 2016, Pal et al. 2019, Bryan et al. 2020, Bryan et al. 2023 ).Additionall y, CM stoc k populations contain sub-populations of genetic v ariants expr essing phenotypic differ ences (Ramsey et al. 2009 ).Inoculation of mice with different plaque-purified clonal isolates fr om these stoc ks r esults in infections of varying severity (Poston et al. 2018 ).Ideally, a clone selected for vaccine studies should reflect the infection profile and pathology of its multiclonal parent stock, because it yields consistent levels of shedding and pathology between individual mice.
The female guinea pig model using the natural pathogen Chlamydia caviae strain provides a second rodent model.Genital tract disease closely resembles that of women, but the lack of imm unological r ea gents is a significant barrier to vaccine studies (Rank and Whittum-Hudson 2010 ).The pig model utilizing Chlamydia suis is being explored as a large animal vaccine model due to the r epr oductiv e cycle and genital tract anatomy being like humans, but r ea gents ar e also less av ailable compar ed to mice and specialized veterinary expertise is often needed due to logistical difficulties associated with infecting large animals (Lorenzen et al. 2015, Amaral et al. 2020 ).Non-human primates continue to serve as major preclinical vaccine testing models prior to phase 1 testing of vaccines in humans .T his challenge model is limited for CT due to ocular and genital tract infections being shorter and self-limited, compared to humans, but provides utility for assessment of vaccine-elicited T and B cell responses to identify correlates of protection.

Correlates of chlamydial immunity
Studies of natural immunity to CT in humans indicate the importance of T cells for CT infection resolution and protection fr om r einfection.Women with confirmed prior infection had lo w er chlamydial loads when reinfected (Batteiger et al. 2010 ).Women who spontaneously cleared infection without treatment exhibited increased CT-specific IFN γ + CD4 T cells and were less likely to be reinfected when compared to women who required antibiotics for infection resolution (Geisler et al. 2013 ).HIV-infected women with low CD4 counts experienced higher rates of chlamydial PID (Kimani et al. 1996 ), and CT-specific IFN γ + CD4 T cells are associated with resistance to reinfection (Russell et al. 2016 ).In contrast, among a cohort of highly CT-exposed women, serum and cervical anti-CT IgG correlated with increased risk of incident infection during a year of follow-up; with hazard ratios increasing 3.6-fold and 22.6-fold with each unit of serum and cervical IgG, r espectiv el y (Darville et al. 2019 ).IFN γ secr eting CD4 T cells (Th1 and Th1/17 cells) are critical players in the protectiv e imm une r esponse to this obligate intr acellular bacterium that replicates within a pr otectiv e v acuole inside epithelial cells (Darville 2021 ).CD4 T cell immunity is enhanced in the presence of chlamydial-specific IgG (Farris et al. 2010 ) by accelerating myeloid cell-mediated killing through opsonization (Naglak et al. 2016 ) and by enhanced Fc-mediated antigen presentation (Moore et al. 2003 ).IFN γ is needed for optimal antibody-mediated imm unity a gainst m urine genital c hlamydial infection (Na glak et al. 2016 ).Evidence for effective in vivo neutralization of chlamydial entry is minimal, potentially reflecting redundancy in adhesinligand interactions.Women with the highest anti-CT IgG responses may be Th2-ske wed, r esulting in inadequate IFN γ responses.Pr e vious data reinforce the importance of eliciting robust and a ppr opriate functional CD4 T cell r esponses, gener ating a milieu that enables antibody-mediated opsonophagocytosis (Naglak et al. 2017 ).Vaccination of male and female mice r e v ealed a similar inter play wher e memory CD4 T cells in females and antibodies in male ejaculate synergized to elicit sterilizing immunity against tr ansmission, suggesting v accination of both sexes could be ideal for reducing infection prevalence in humans (O'Meara et al. 2016 ).
Short-liv ed sterilizing imm unity after infection is observ ed in animal models (Rank and Whittum-Hudson 2010 ).Geneknoc k out, depletion, and adoptive transfer studies in mice consistentl y demonstr ate that CD4 T cells ar e the primary mediators of immunity (Perry et al. 1997, Wang et al. 1999, Stary et al. 2015 ) and that polyfunctional Th1 cells (IFN γ + TNF α+ interleukin (IL)-2 + ) secreting high cytokine levels correlate with protection (Yu et al. 2011, Poston et al. 2017 ).These studies have focused on imm une r esponses in the female r epr oductiv e tr act, but male mouse studies have also demonstrated the importance of CD4 T cells in clearing infection (Cunningham et al. 2010, Sobinoff et al. 2015, O'Meara et al. 2016 ).Most activated CD4 T cells are not terminall y differ entiated and may shift between subsets to alter their cytokine profile (i.e .T h17-T h1 shift) or exhibit properties of multiple subsets where a more extensive range of cytokines are produced (i.e.bifunctional Th1/17) (Zielinski et al. 2012, Wacleche et al. 2016 ).Th1 or Th1/17 cells produce IFN γ that induces cellular responses that starve intracellular chlamydiae of tryptophan (Thomas et al. 1993, Nelson et al. 2005, Leonhardt et al. 2007 ) and enhance phagocyte effector functions (Al-Zeer et al. 2013, Tietzel et al. 2019 ).IL-17A independentl y pr omotes epithelial integrity and induces the production of antimicrobial peptides (Mills 2023 ).Vaccine-induced IL-17A is important for protection against other mucosal bacterial pathogens (Lin et al. 2010 ) and higher levels of IL-17 induced by a subunit outer membr ane pr otein v accine have been linked to lo w er chlamydial bur den in mice and less disease upon challenge (Lizarraga et al. 2020 ).In CT-exposed women, Th17-lineage cells were significantly increased in those who did not experience follow-up CT infection compared to those who did, suggesting an important role for these cells in protection (Yount et al. 2023 ).
The role of CD8 T cells against Chlamydia infection is less clear.Mouse models r e v eal they ar e dispensable for clear ance.Yet, adoptiv el y tr ansferr ed c hlamydia-specific CD8 T cell clones can migrate to the infected genital tract and enhance clearance through the production of IFN γ (Igietseme et al. 1994 ).Human studies hav e corr elated the fr equency of antigen-specific CD8 T cells with limiting infection to the cervix (Russell et al. 2016 ).This could implicate a role in preventing ascension to the upper genital tract and the de v elopment of disease.Ho w e v er, the pr oduction of TNF by Chlamydia -specific CD8 T cells during infection of naïve mice has been shown to play a role in immunopathology (Murthy et al. 2011, Vlcek et al. 2016 ).Recent data r e v eal this may be a phenomenon restricted to the intravaginal challenge of naïve mice since vaccine-elicited antigen-specific CD4 T cells were able to prevent this pathological response (Murthy et al. 2024 ).
T cells can tr affic fr om the circulation or respond directly in the infected tissue as a tissue-resident memory (TRM) population.Adjuv ants and r outes of imm unization that induce Th1 or Th1/17 cells that traffic to the genital tract will promote vaccine efficacy.Intr anasal v accination of female mice with inactivated Chlamydia complexed to nanoparticles with the toll-like receptor (TLR)7/8 agonist resiquimod induced IFN γ + TRM CD4 T cells in the genital tract (Stary et al. 2015 ).Mice vaccinated subcutaneousl y wer e unpr otected despite gener ating a systemic T cell r esponse, demonstrating an essential role for non-circulating TRM in mice.A pr e v ailing hypothesis is that m ucosal imm unization or infection induces plastic Th17 cells that migrate to infected tissue where they differentiate into Th1 (ex-Th17) or Th1/17 cells that become persistent tissue-resident cells capable of responding r a pidl y to r einfection (Amezcua Vesel y et al. 2019 ).The female genital tract (FGT) is a toler ogenic envir onment that r estricts T cell influx in the absence of inflammation and differs from other m ucosal sites, suc h as the lung, by the absence of secondary lymphoid tissue (Nakanishi et al. 2009 ).Instead, memory lymphocyte clusters, consisting of CD4 T and B/plasma cells, have been observed in murine uterine tissue months after the infection resolves (Morrison and Morrison 2000 ).Inducing or boosting these immune populations by mucosal vaccination may be necessary for optimal immunity to CT .Most of the r esearc h defining correlates of Chlamydia immunity has focused on the female r epr oductiv e tr act and it is not definitiv el y known if these findings dir ectl y translate to other clinically relevant sites like the ocular conjunctiv a, r ectum, and male ur ethr a/testis .T he evidence to date suggests that these correlates likely translate to other mucosal sites.Ho w e v er, additional testing to confirm this translatability in large animal models like pigs or non-human primates would be ideal.
Murine CD4 T cell clones hav e demonstr ated m ultiple mec hanisms of CM inhibition.These include nitrous oxide production, perforin-mediated cytolysis, and T-cell degranulation after major histocompatibility complex (MHC)-II-dependent recognition of infected epithelial cells (J ay ar a pu et al. 2010 ).Further investigation r e v ealed an inducible nitric o xide synthase (iNOS)-de pendent mechanism and an independent mechanism of clearance that requires Plac8 (Johnson et al. 2012 ).It is unknown if similar mechanisms occur in humans.Both CM and CT are susceptible to the effects of IFN γ but hav e e volv ed differ ent mec hanisms for e v asion.Human IFN γ activates indoleamine 2,3-dioxygenase that converts tryptophan to N-form yl-kyn urenine thereby depleting tryptophan necessary for chlamydial replication (Byrne et al. 1986, Beatty et al. 1993 ).CT genital strains have evolved to evade this mechanism by encoding a functional tryptophan synthase to conv ert indoles pr oduced by other micr obiota into tryptophan to support their intracellular replication (Fehlner-Gardiner et al. 2002 ).Murine IFN γ induces expression of p47 GTPases in murine cells that restrict CT growth (Nelson et al. 2005, Bernstein-Hanley et al. 2006 ).CM resistance to murine IFN γ may be due to the function of the putative inclusion membrane protein TC0574 that could inhibit inclusion destruction by caspases or related pro-death cysteine proteases (Giebel et al. 2019 ).Humans lack p47 GTPases but possess IFN γ inducible guanylate-binding proteins (GBP) that possess GTPase activity.Data suggest that CT can block ubiquitination by GBPs but remain susceptible to GBP-mediated inflammasome activation (Finethy et al. 2015 ).

Platform selection
The first chlamydial vaccines in the 1960s used live or formalinfixed whole bacteria to pr e v ent ocular infection.These studies demonstr ated short-liv ed pr otection fr om activ e tr ac homa (Poston et al. 2019 ).Similar results were obtained using high doses in non-human primates (Kari et al. 2011 ).There has been recent success in removing pathogenic factors from Chlamydia spp. with the goal of optimizing whole-cell-based vaccines .T he plasmid encoded pgp3 and c hr omosomal TC0237/TC0668 were identified as virulence factors that induce hydrosalpinx in CM-challenged mice (Liu et al. 2014, Chen et al. 2015, Huang et al. 2015, Conrad et al. 2016 ).Str ains lac king these genes induced a pr otectiv e memory response in the absence of oviduct pathology consistent with previous studies using plasmid-deficient CM (O'Connell et al. 2007 ).T hese data pro vide valuable insight into chlamydial pathogenicity but are not likely to overcome safety concerns for use as human vaccines.
The use of inactivated CT EBs may alleviate some safety concerns but has not demonstrated significant protection in mouse models.Although UV-inactivated whole organisms have been used to investigate the induction of protective responses, there are concerns regarding cost, safety, lack of scalability, reproducibility, and poor efficacy in earl y c hlamydial tr ac homa pr otection trials (Grayston et al. 1963, So w a et al. 1969, Grayston and Wang 1978 ) that preclude their use as commercial vaccines .T he short-term pr otection observ ed implies an effectiv e v accine is feasible with the goal of a safe subunit vaccine.Of candidate immunogens that ha ve been in vestigated to date, the major outer membrane protein (MOMP) is the most studied.Vaccination of female mice with nativ e MOMP isolated fr om the c hlamydial env elope pr ovided significant protection against challenge, determined by lowered cervical burden, acceler ated clear ance, and r educed oviduct pathology (P al et al. 2005(P al et al. , Farris et al. 2010 ).Ho w e v er, these r esults did not translate with recombinant MOMP (Sun et al. 2009, Tifrea et al. 2011, Pal et al. 2017 ), suggesting that some of the protection observed was deriv ed fr om MOMP-conformation-dependent antigen r ecognition.This confirmation dependency could impact both memory B and T cell induction (Tifrea et al. 2011, Peng et al. 2022 ).Antibody recognition of conformational epitopes may promote combined neonatal Fc r eceptor tr anslocation to the subepithelium and antigen-presentation by Fc-receptor expressing professional antigen-pr esenting cells (Moor e et al. 2003, Armita ge et al. 2024 ).This would accelerate a memory CD4 T cell response and may be important for optimal early control and reduced upper genital tract pathology.The T cell response may also be dir ectl y affected by antigen conformation.The le v el of Yersinia pestis Caf1 antigen processing and presentation was dependent on the structure of MHC class II binding epitopes (Musson et al. 2006 ).Epitopes in the globular domain were presented by MHC class II after lysosomal processing, while epitopes in the flexible strand were presented without pr oteol ytic pr ocessing.T his could ha ve implications for MOMP epitope presentation due to the cysteine residues that form intramonomeric disulfide bonds in the tr ansmembr ane r egion (Yen et al. 2005 ).T hus , trimeric native MOMP and monomeric recombinant MOMP may load different MHC class II epitopes that could influence the breadth and magnitude of the CD4 T cell response.
A human vaccine with conformationally correct MOMP isolated from live bacteria is not amenable to upscaling for commercial production and would be complicated by multiple serovars expr essing differ ent MOMP extr acellular v ariable domains.Attempts to fold r ecombinantl y expr essed MOMP in lipid nanodiscs were successful in demonstrating efficacy in an animal model (Tifrea et al. 2021 ).Ho w ever, endotoxin contamination remains a concern for nanoparticle v accine form ulations and makes manufacturing and scale-up problematic (Hannon andPrina-Mello 2021 , Costa et al. 2023 ).An a ppr oac h to bypass the need for conformationall y corr ect r ecombinant MOMP has been tested in a phase 1 study of CTH522, consisting of MOMP imm unor epeat segments spanning the variable domain four region of serovars D, E, F, and G and adjuvanted with liposomal cationic adjuvant formulation (CAF01) or aluminum hydroxide (alum) (Abraham et al. 2019 ).These form ulations wer e deliv er ed thr ee times via intr amuscular injection follo w ed b y tw o unadjuv anted intr anasal inoculations and were well tolerated and immunogenic in humans.Disa ppointingl y, significant pr otection was not observ ed after the administration of the CTH522/CAF01 vaccine to nonhuman primates (Lorenzen et al. 2022 ).
Other candidate membrane antigens tested in preclinical vaccine studies include pol ymor phic outer membr ane pr oteins (Yu et al. 2014 ) and the adhesin, outer membrane complex B (Olsen et al. 2010, Finco et al. 2011 ) but none has ac hie v ed the le v el of protection induced by native MOMP.When recombinant Chlamydia protease-like activity factor (CPAF), a secreted protein important in pathogenesis (P asc hen et al. 2008 ), was paired with IL-12 or CpG oligodeo xyn ucleotides (ODN) (TLR9 agonist) and deli v er ed intr anasall y, it led to an abbr e viated infection and reduced oviduct pathology in mice (Cong et al. 2007, Murthy et al. 2007 ).Profiling of human antibody responses revealed this antigen to be immunodominant and imm unopr e v alent in Chlam ydia -exposed women (Liu et al. 2022 ).These data indicate this highly conserved antigen is a strong candidate for a human vaccine that may be paired with membrane proteins like MOMP , PmpF , and/or PmpG (Yu et al. 2012, Yu et al. 2014 ).
Most clinicall y av ailable v accines a gainst bacterial pathogens induce antibodies as a primary mode of protection.Ho w ever, c hlamydial pr otection is dependent on CD4 T cell-mediated responses (Perry et al. 1997, Wang et al. 1999, Yu et al. 2011, Stary et al. 2015, Poston et al. 2017 ).Until the past decade, generating vaccines against intracellular bacterial pathogens, where cellmediated immunity is required for protection, was hampered by the lack of safe and effective Th1 and Th17-inducing adjuv ants (Lav elle and Ward 2022 ).Char ge-switc hing synthetic adjuv ant particles incor por ating the TLR7/8 a gonist R848 (r esiquimod) complexed to UV-inactivated chlamydial EBs induced significant pr otection fr om burden and disease upon v a ginal c hallenge in a female mouse model, demonstrating the po w er of adjuvants to accelerate induction of robust chlamydial-specific IFN γ + T cell responses (Stary et al. 2015 ).Early studies using CPAF as an imm unogen demonstr ated pr otection when CPAF was adjuv anted with IL-12 or multiple doses of CpG before and after CPAF immunization (Cong et al. 2007, Murthy et al. 2007 ).Ho w e v er, the toxicity of IL-12 administration and the implausibility of delivering CpG on consecutive days make these a ppr oac hes unsuitable for human vaccination.
Adjuvants that target endosomal TLR7 and TLR9 pathways initiate the production of IL-12 and IFNs that induce IFN γ + CD4 T cells (Wille-Reece et al. 2005, Kastenmuller et al. 2011, Temizoz et al. 2015, Carroll et al. 2016 ).A single adjuvant may be unable to elicit a broad enough cytokine response sufficient to driv e pr otectiv e T cell-mediated immunity, but two or more agonists can syner gisticall y enhance v accine efficacy (Temizoz et al. 2015, Kocabas et al. 2020 ).Stim ulator of interfer on genes (STING), induces the production of cytokines that drive cellmediated immunity (Ahn and Barber 2019 ).Agonists of the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-STING pathwa y ha v e emer ged as safe adjuv ants that activate type I IFNs, pro-inflammatory cytokines, and the inflammasome for IL-1 β and IL-18 production (Webster et al. 2017, Ou et al. 2021 ).These agonists elicit germinal center formation and mor e potent humor al r esponses when compar ed to adjuv ants like CpG and shift the response from Th2/IgG1 dominant to a balanced IgG1/IgG2 (Th1) profile (Gray et al. 2012 ).STING is dir ectl y activ ated b y c yclic dimeric adenosine monophosphate (cdi-AMP, CDA), a bacterial metabolite.CDA matures dendritic cells and promotes antigen capture, processing, and presentation for T cell priming, resulting in T h1 and T h1/17 responses (Ebensen et al. 2011, Volckmar et al. 2019 ).STING a gonists hav e not been tested as v accine adjuv ants a gainst CT but hav e enhanced host r esistance a gainst SARS-CoV-2, Mycobacterium tuberculosis, and Bordetella pertussis , when administered via parenteral and mucosal r outes (Carr oll et al. 2016, Allen et al. 2018, Van Dis et al. 2018, Humphries et al. 2021 ).STING agonists synergize with TLR7 or TLR9 agonists for broader cytokine responses, leading to higher frequencies of antigen-specific IFN γ + CD4 T cells (Temizoz et al. 2015, Collier et al. 2018, Kocabas et al. 2020, Zhang et al. 2022 ).A phosphorothioate CDA analog, 2'3'-c-di-AM(PS)2 (Rp,Rp) (ADU-S100), has increased in vivo stability and stimulates human and mouse STING alleles (Corrales et al. 2015 ) and was safe in clinical trials (Meric-Bernstam et al. 2022 ).The non-n ucleotid yl small molecule diABZI had demonstrated increased potency and safety in humans making it one of the most promising STING agonists to date (Ramanjulu et al. 2018, Talley et al. 2021 ).
Vaccination with CTH522 plus CAF01 induced higher cellmediated IFN γ responses and more consistent serum antibody titers than CTH522 adjuvanted with alum.CAF01 contains liposomes formed by N ,N -dimethyl-N ,N -dioxtadecylammonium (DDA) with the synthetic mycobacterial immunomodulator α,αtrehalose 6,6 -dibeheneate (TDB).DDA targets the vaccine antigen to antigen-presenting cells and the TDB induces cytokines that drive a Th1/17 response (Hansen et al. 2008, Wern et al. 2017 ).This response was demonstrated to be MyD88-dependent but TLR-independent (Agger et al. 2008 ).Later studies have demonstrated that TDB initiates the inflammatory response through the macropahge inducible Ca2 + -dependent lectin receptro (Mincle) receptor and triggers Syk-Card9-dependent APC activation along with the IL-1/MyD88 pathway (Werninghaus et al. 2009, Schoenen et al. 2010, Desel et al. 2013 ) leading to interferon alpha/beta r eceptor alpha c hain (IFNAR1)-dependent CD4 T cell r esponses (McEntee et al. 2020 ).The safety of Mincle agonists in human trials (van Dissel et al. 2014, Abraham et al. 2019, Dejon-Agobe et al. 2019 ) is encour a ging, but the contribution of these responses to protection is not known since a phase II trial has yet to begin.Testing clinically safe Mincle, TLR, and STING agonists alone and in combination may provide a path forw ar d to an effective CT vaccine.
Antigen-adjuvant conjugates, or immunogens coupled to synthetic adjuvants, further potentiate the immune response by codeliv ering v accine components to an antigen-pr esenting cell, pr oviding prolonged antigen presentation that induces adaptive imm une r esponses thr ough intr acellular stor a ge and depot effects, and boosting production of immunostimulatory cytokines and c hemokines (v an Montfoort et al. 2009, Moyle 2017, Xu and Moyle 2018, Ho et al. 2021 ).(Fig. 1 ) This a ppr oac h has superior efficacy in preclinical vaccination studies (Tighe et al. 2000, Wille-Reece et al. 2005, Wille-Reece et al. 2005, Kastenmuller et al. 2011, Zom et al. 2018, Bha gc handani et al. 2021 ).Furthermore, conjugating these agonists to vaccine antigens facilitates endosomal uptake and increases TLR signaling for APC activation, which reduces the amount of adjuvant required for immunogenicity and lo w ers reactogenicity (Oh and Kedl 2010, Irie et al. 2020, Weiss et al. 2021, Zhang et al. 2022 ).Futur e v accine str ategies could explor e the incor por ation of TLR7/9, Mincle, and STING agonists alone and in combination with adsorption or bioconjugation strategies to further enhance immunogenicity by upregulating inflammatory cytokines and type I interferons.
Vir al v ectors ar e an efficacious deliv ery system for imm unization that provides expression of antigens within target cells.These v ectors ar e usuall y liv e-attenuated or r eplication-deficient, which allows for the preservation of immunogenicity while reducing pathogenicity.They hav e pr ovided a pr oof-of-concept a ppr oac h for pr eclinical assessment of putativ e antigens and profiling correlates of immune protection.Some viral vector vaccines that hav e pr ogr essed to clinical trials and/or licensing include recombinant canarypox (ALVAC-Human immunodeficiency virus), Modified Vaccinia Ankara (MVA)-E2 (human pa pillomavirus), r ecombinant vesicular stomatitis (VSV)-Zaire ebola virus (ZEBOV), chimpadenovirus (ChAdOx1), and recombinant Ad26 for respiratory syncytial virus (RSV) and Zika virus (ZIKV) infections (Rerks-Ngarm et al. 2009, Rosales et al. 2014, Dahlke et al. 2017, Williams et al. 2020, Ewer et al. 2021, Pollard et al. 2021, Salisch et al. 2021 ).Vir al v ector v accines hav e the benefit of inducing a broad ada ptiv e imm une r esponse c har acterized by high frequencies of CD4 T cells, cytotoxic CD8 T cells (CTLs), and class-switched antibody and memory B cells .Recombinant adeno viruses ha ve demonstrated the most utility to date due to their high transduction efficiency, antigen expression, and broad tropism.These v ectors hav e been licensed as SARS-CoV-2 vaccines and include Vaxze v eria (ChAdOx1 nCoV-19/AZD1222), Janssen COVID-19 vaccine (Ad26.COV2.S), and Sputnik V (Gam-COVID-Vac).MVA is also a promising vector derived from the highly attenuated Ankara str ain.This v ector is licensed as a v accine for smallpox and has demonstrated safety and efficacy in human trials (McShane et al. 2004, de Vries et al. 2018, Chiuppesi et al. 2022 ).It has commonly been used in heterologous prime-boost regimens with adenoviral v ectors a gainst m ultiple pathogens (Rampling et al. 2016, Tapia et al. 2016 ) and the heterologous combo of MVA BN-Filo and Ad26-ZEBOV is licensed for human use against Ebola virus disease (Pollard et al. 2021 ).
Vir al v ector v accines pr ov en safe in humans provide an opportunity to test recombinant chlamydial antigens using this plat-form due to their ability to induce Th1 r esponses.To date, ther e are limited studies investigating viral vectored chlamydial vaccines in animal models.Intranasal delivery of influenza-vectored MOMP epitopes inserted into the neuraminidase stalk region induced durable MOMP-specific Th1 responses in the spleen and dr aining l ymph nodes (He et al. 2007 ).This was associated with reduced burden after intravaginal challenge with CT serovar D. Intr anasal administr ation of adenovirus-v ector ed CP AF (AdCP AF) elicited a balanced CPAF-specific IFN γ response between CD4 and CD8 T cells in C57BL/6, C3H/HeN, and BALB/c mice after a single dose (Brown et al. 2012 ).Mice receiving a heterologous regimen with AdCPAF and r ecombinant CPAF adjuv anted with CpG and the immunomodulatory peptide HH2 demonstrated a greater Th1 r esponse compar ed to a mixed Th1/17 response following homologous rCPAF prime-boost.Both prime-boost r egimens r educed the fr equency of hydr osalpinx by 50% in C3H/HeN mice compared to single-dose AdCPAF administration.The use of hAd5-MOMP and MVA-MOMP in a prime-boost strategy also incor por ating DNA-MOMP and recombinant MOMP plus CAF01 has recently been investigated in cynomolgus macaques (Lorenzen et al. 2022 ).The regimen utilizing DNA-MOMP as the priming dose follo w ed b y CAF01 adjuvanted CTH522 elicited a more balanced CD4/CD8 response and abbr e viated the duration of infection.
The use of mRNA technology as a vaccine platform has made major strides .T he mRNA that encodes the selected immunogen is encapsulated in lipid particles for protection against degradation and delivery into host cells where the mRNA is translated into protein.This mRNA and liposome combination provides an intrinsic adjuvant effect, allows for repeated administration, and avoids pre-existing immunity associated with some viral vectors (Verbeke et al. 2022, Xie et al. 2023 ).This platform has consistently demonstrated safety in humans, comes with no risk of insertional m uta genesis, and has the benefit of r a pid, low-cost manufacturing.This was r ecentl y demonstr ated with the r a pid turnar ound of the Pfizer Tozinameran (BNT162b2) and Moderna mRNA-1273 v accines a gainst SARS-CoV-2.These v accines wer e pr oduced in an in vitro cell-free transcription reaction that expedites the conventional vaccine manufacturing process.Both were the first mRNA vaccines licensed for humans and generated both neutralizing antibodies and Th1-biased responses (Polack et al. 2020, Baden et al. 2021 ).This platform has been effectiv el y utilized against SARS-CoV-2 with additional vaccines against influenza, RSV, and ZIKV in clinical trials (Feldman et al. 2019, Aliprantis et al. 2021, Essink et al. 2023 ).Pr eclinical r esearc h studies ar e inv estigating the efficac y of mRN A v accines a gainst CT and other bacterial pathogens (Maruggi et al. 2017, Kon et al. 2023, Pine et al. 2023 ).

Route of immunization
Curr ent FDA-a ppr ov ed m ucosal v accines use inactiv ated or liv eattenuated pathogens with only one intranasal vaccine to date (FluMist ®) with the rest targeting gut pathogens and are deliver ed or all y.Subunit v accines ar e safe , stable , and highly manufacturable , but ha ve exhibited poor immunogenicity when given intr anasall y mainl y due to the challenges of delivery.Vaccine uptake in the underlying mucosal immune compartment is hindered by pr oteol ytic enzymes , acidic conditions , m ucociliary clear ance, and the lack of diffusion across the epithelial monola yer.T hese are barriers to effective antigen delivery to the lamina propria and dr aining l ymph nodes wher e dendritic cells ca ptur e and pr esent antigens to CD4 T cells.(Fig. 2 ) Priming of CD4 T cells in the nasalassociated lymphoid tissue imprint chemokine receptors and integrins like CCR10 and α4 β1 that allow effector CD4 T cells to home to both the r espir atory and genitourinary tracts (Holmgren and Czerkinsky 2005 ).Intr anasal v accination using TLR agonistadjuv anted inactiv ated Chlam ydia ac hie v ed superior genital tr act protection in mice compared to parenteral routes (Murthy et al. 2007, Stary et al. 2015 ).Every chlamydial vaccine tested preclinicall y has ac hie v ed superior r esults with m ucosal administr ation when compared directly to systemic administration (de la Maza et al. 2021 ).Ho w e v er, the field has lac ked safe m ucosal adjuv ants until r ecentl y.Man y adjuv ants ar e too r eactogenic when deliver ed systemicall y or cause nasal irritation.Ho w e v er, the issues of reactogenicity and delivery may be overcome with sublingual (s.l.) delivery using tablets (Kelly et al. 2022 ) or thermor esponsiv e gels (White et al. 2014, Lal et al. 2017 ) Mucosal vaccination offers advantages over systemic immunization that include avoiding needle fears, the potential for selfadministration, and induction of mucosal and systemic immunity.

Clinical testing
The field seeks to de v elop an efficacious CT vaccine to prevent male and female genital tract disease with the larger goal of preventing sexual transmission.A vaccine would ideally target adolescents before sexual debut to establish immunity prior to exposure.Human clinical trials for efficacy will require the enrollment of sexually active adults with a target age of 18-25 years since CT is most pr e v alent in this a ge gr oup (Miller et al. 2004 ).Both cis-and transgender males and females would be eligible for enrollment, and efficac y w ould be assessed b y nucleic acid amplification testing (NAAT) of clinically obtained samples.Reductions in the frequency of infected individuals and bacterial burden in those who become infected could serve as primary endpoints in a clinical trial.Based on modeling studies, a CT vaccine that achieves at least a 50% reduction in the rate of infection for 10 years will decr ease infection pr e v alence by 50% after 13 years and essentially eradicate the infection in 24 years (de la Maza and de la Maza 1995 ).A high-cov er a ge v accine that r educes the c hlamydial peak load by 2-log 10 could er adicate a c hlam ydial e pidemic in ∼20 years (Gray et al. 2009 ).Thus, a 50% effective vaccine that results in a 2-log 10 reduction in burden among those who acquire infection r elativ e to individuals in the unvaccinated arm would be highly beneficial by decreasing the prevalence of infection and reproductive morbidities like PID and infertility.
The use of r epr oductiv e tr act disease as a secondary clinical endpoint is influenced by multiple factors .T he clinical diagnosis of PID is insensitive and nonspecific with multiple causes.Although CT causes ∼one-third of PID cases, determining causation would pr ov e difficult, and infertility may not be recognized for years after prior CT infection(s).The pr e viousl y executed la par oscopic dia gnosis is inv asiv e and no longer r outinel y performed.Determining more precise and practical measures of upper genital tract inflammation and disease is a priority for the design of practical clinical trials.Future work will seek to determine the role of techniques like MRI and po w er Doppler along with the potential for blood biomarkers for less inv asiv e sampling (Zheng et al. 2018, Zheng et al. 2018 ).Endometrial biopsies are a minimally invasive procedure used to determine upper genital tract infection and inflammation.Ho w ever, a high cervical bacterial burden may be an acceptable surrogate for upper genital infection (Russell et al. 2016 ).A non-inv asiv e biomarker for upper genital tract ascension would be a powerful tool to use in conjunction with NAAT testing.This tandem a ppr oac h would help determine if a decrease in infection parallels a decrease in disease and determine if PID is pr e v ented in v accinated individuals who become infected.It would also address if br eakthr ough infection led to an enhanced risk for PID.This could also have important implications in determining vaccines that may enhance disease .For example , vir al v ector v accines with the potential to elicit high frequencies of antigen-specific CD8 T cells, which may not effectiv el y r educe Chlam ydia burden, could potentiall y enhance immunopathology because of ongoing stimulation during chronic infection in the absence of pr otectiv e CD4 T cells.Futur e r esearc h aims to address better ways of measuring upper genital tract infection, inflammation, and disease for the purpose of clinical vaccine testing.

Conclusion
Major pr ogr ess has been made in Chlam ydia v accinology to identify the importance of CD4 T cell immunity and the potential for resident memory populations to enhance protection.Recent advancements in mucosal adjuvant development provide a path forw ar d for de v eloping a v accine that is safe and well-tolerated in humans.Pre-clinical testing of these nov el v accine form ulations in mice will help determine efficacy and safety against genital infection and disease .T his will help triage vaccines for further testing in non-human primates prior to Phase 1 clinical trials.A partiall y pr otectiv e v accine would significantl y r educe infection pr e v alence.Curr ent r esearc h efforts will continue to identify vaccines suitable for human translation and identify correlates of pr otectiv e imm unity v ersus pathogenic r esponses.

Figure 2 .
Figure 2. Intranasal and sublingual immunization activates submucosa dendritic cells (left) that migrate to the draining cervical lymph nodes and activate antigen-specific CD4 T cells (right) that provide transmucosal immunity.
. These formulated sublingual vaccines enhance contact time between antigen and the or al m ucosa and s.l.deliv ery of MOMP adjuv anted with CTA1-DD (fusion protein of cholera toxin A1 subunit and immunoglobulin binding domain from Staphylococcus aureus protein A) led to a reduced incidence of oviduct pathology in mice challenged with CM (O'Meara et al. 2014 ).Multiple deliv ery a ppr oac hes ar e being explored, opening the door for the practical development of mucosal delivery via intranasal and sublingual routes (Huang et al. 2022 ) that provide genital imm unity.Or al imm unization with liv eattenuated Chlamydia (Wang et al. 2018 , Zhou et al. 2022 , Wang et al. 2023 ) and a MOMP v accine adjuv anted with c holer a toxin, CpG-ODN, and Lipid C have also demonstrated protection in mice (Hick e y et al. 2010 ).Covalently coupling protein antigens with FcRn-binding moieties or in liposomes may facilitate better transportation across the mucosal epithelium resulting in enhanced antigen-specific T-and B-cell responses (Lu et al. 2011 , Ye et al. 2011 , Olsen et al. 2015 , Tada et al. 2018 , Kumar et al. 2021 , Ochsner et al. 2021 , Hartwell et al. 2022 , Li et al. 2023 , Ozberk et al. 2023 ).